Ions move across plasma membranes and organelle membranes through channels created by proteins, which allows for the formation of concentration gradients between exterior and interior compartments. Ion channels participate in, and regulate, cellular processes as diverse as the generation and timing of action potentials, energy production, synaptic transmission, secretion of hormones and the contraction of muscles. In fact, many drugs exert their specific effects via modulation of ion channels. Examples include antiepileptic compounds like phenyloin and lamotrigine, which block voltage-dependent sodium channels in the brain, antihypertensive drugs like nifedipine and diltiazem, which block voltage-dependent calcium channels in smooth muscle cells, and stimulators of insulin release like glibenclamide and tolbutamide, which block ATP-regulated potassium channels in the pancreas.
There are many types of ion channels including, for example, ligand-gated channels, which open or close in response to the binding of signalling molecules; cyclic nucleotide-gated channels, which open in response to internal solutes and mediate cellular responses to second messengers; Stretch-activated channels, which open or close in response to mechanical forces that arise from local stretching or compression of the membrane; G-protein-gated channels, which open in response to G protein-activation via its receptor; and voltage-gated channels, which open or close in response to changes in the charge across the plasma membrane.
Finding new drugs which have specific modulatory effects on ion channels requires methods for measuring and manipulating the membrane potential and/or concentration gradient of cells with the ion channels present in the membrane. A number of methods exist that can be used to measure cell transmembrane potentials and/or concentration gradients and to measure the activities of specific ion channels. For example, patch-clamp recording was the first technique capable of monitoring the function of single biological molecules by measurement of single-channel currents (Neher, E. and B. Sakmann, “Single-channel currents recorded from membrane of denervated frog muscle fibres,” Nature (London), 260:799-802 (1976); Hamill, O. P. et al., “Improved patch-clamp techniques for high-resolution current recording from cells and cell-free membrane patches,” Pflugers Arch., 391:85-100 (1981)). Using the patch-clamp technique, the properties of ion channels can be studied by means of a very fine pipette (with an opening of about 0.5 μm) that is pressed against the plasma membrane of either an intact cell or that is used to pull away the plasma membrane from the cell and the preparation placed in a test solution of desired composition. In so doing, current flow through a single ion channel can then be measured. Techniques are known in the art for performing patch-clamp techniques that are suitable (Petersen, O. H. et al., “The Patch-Clamp Technique: Recording Ionic Currents Through Single Pores in the Cell Membrane,” Physiology, 1(1):5-8 (1986); Boulton, A. A. et al., Patch-Clamp Applications and Protocols, Vol. 26 (1995)). For example, common techniques may include performing cell-free ion-channel recording, the whole-cell patch clamp technique, concentration clamp technique, the pressure clamp method, the perfusion of patch clamp electrodes, loose patch-clamp technique, single-channel recording and the perforated patch-clamp technique. However, a major limitation of the patch clamp technique as a general method in pharmacological screening is its low throughput. Typically, a single, highly trained operator can test fewer than ten compounds per day using the patch clamp technique. Furthermore, the technique is not easily amenable to automation, and produces complex results that require extensive analysis by skilled electrophysiologists.
The use of optical detection systems provides for significantly greater throughput for screening applications and advances in optical techniques have allowed direct visualization of calcium signaling at the cellular and subcellular level (Lino, R. et al., “Single molecule imaging of green fluorescent proteins in living cells: E-cadherin forms oligomers on the free cell surface,” Biophys. J., 80:2667-2677 (2001); Schnitzer, M. J. et al., “Force production by single kinesin motors,” Nat. Cell Biol., 10:718-723 (2000); Sonnleitner, A. et al., “Structural rearrangements in single ion channels detected optically in living cells,” Proc. Natl. Acad. Sci. U.S.A., 99:12759-12764 (2002); Zou, H. et al., “Imaging Ca21 entering the cytoplasm through a single opening of a plasma membrane cation channel,” J. Gen. Physiol., 114:575-588 (1999); Zou, H. et al., “Visualization of Ca21 entry through single stretch-activated cation channels,” Proc. Natl. Acad. Sci. U.S.A., 99:6404-6409 (2002); Wang, S. Q. et al., “Ca21 signalling between single L-type Ca21 channels and ryanodine receptors in heart cells,” Nature (London), 410:592-596 (2001); Demuro, A. and I. Parker, “Optical single channel recording: imaging Ca21 flux through individual N-type voltage-gated channels expressed in Xenopus oocytes,” Cell Calcium, 34:499-509 (2003); Demuro, A. and I. Parker, “Imaging the activity and localization of single voltage-gated Ca21 channels by total internal reflection fluorescence microscopy,” Biophys. J., 86:3250-3259 (2004).) Indeed, advances in the development of video imaging and confocal microscopy, have led to the discovery of polarized, subcellular calcium signals in various cell types (Knot, H. J. et al., “Twenty Years of Calcium Imaging: Cell Physiology to Die For,” Mol. Interv. 5:112-127 (2005)). The shape of intracellular calcium signals (i.e., amplitude and frequency) is determined by the distribution of calcium-releasing channels and mechanisms that limit calcium elevation (Jiang, Y. et al., “Numerical Simulation of Ca2+ ‘Sparks’ in Skeletal Muscle,” Biophys J 77(5): 2333-2357 (1999)). In addition, development of new cell permeable fluorescent reporters, such as luminescent photoproteins, fluorescent proteins and fluorescent dyes, has opened the way for dynamic cellular assays by allowing activity at drug targets to be determined in living cells (Zhang, J. et al., “Creating New Fluorescent Probes for Cell Biology,” Nature 3:906-918 (2002)). For example, one optical method of analysis has been previously described (Gonzalez and Tsien, “Improved indicators of cell membrane potential that use fluorescence resonance energy transfer,” Chemistry and Biology, 4(4):269-277 (1997); Gonzalez and Tsien, “Voltage sensing by fluorescence resonance energy transfer in single cells,” Biophysical Journal, 69:1272-1280 (1995); and U.S. Pat. No. 5,661,035), that comprises two reagents that undergo energy transfer to provide a ratiometric fluorescent readout that is dependent upon the membrane potential. The ratiometric readout provides important advantages for drug screening including improved sensitivity, reliability and reduction of many types of experimental artifacts.
As currently practiced in the art, drug discovery is a long and multiple step process involving identification of specific disease targets, development of an assay based on a specific target, validation of the assay, optimization and automation of the assay to produce a screen, high throughput screening of compound libraries using the assay to identify “hits”, hit validation and hit compound optimization. The output of this process is a lead compound that goes into pre-clinical and, if validated, eventually into clinical trials. In this process, the screening phase is distinct from the assay development phases, and involves testing compound efficacy in living biological systems.
Bioinformatics, genomics, proteomics and high throughput screening have become indispensable in identifying potential new drug targets, predicting drug interactions, and increasing capacity and efficiency in the areas of target identification. However, even with these developing technologies, there is a need to measure multi-dimensional information from cells and a need for tools that provide increased information handling capability. These aspects of drug discovery make the observation of ion fluctuation particularly suitable to measuring multiple parameters of cell response to compound administration. Indeed, optical imaging methods for screening large numbers of compounds are known in the art (See, e.g., U.S. Pat. No. 6,875,578).
The conventional measurement in early drug discovery assays was radioactivity. However, the need for more information, higher throughput and miniaturization has caused a shift towards using fluorescence detection. Fluorescence-based reagents can yield more powerful, multiple parameter assays that are higher in throughput and information content and require lower volumes of reagents and test compounds. Fluorescence is also safer and less expensive than radioactivity-based methods. The types of biochemical and molecular information now accessible through fluorescence-based reagents applied to cells include ion concentrations, membrane potential, specific translocations, enzyme activities, gene expression, as well as the presence, amounts and patterns of metabolites, proteins, lipids, carbohydrates, and nucleic acid sequences (DeBiasio, R. L. et al., “Myosin II transport, organization, and phosphorylation: evidence for cortical flow/solution-contraction coupling during cytokinesis and cell locomotion,” Mol. Biol. Cell., 7(8):1259-82 (1996); Heim, R. and Tsien, R. Y., “Engineering green fluorescent protein for improved brightness, longer wavelengths and fluorescence resonance energy transfer,” Curr. Biol. 6(2):178-82 (1996)).
Conventional means of imaging fluorescent samples provide calculations of total fluorescence average over a cell sample. For example, Science Applications International Corporation (SAIC) (Seattle, Wash.) describes an imaging plate reader that uses a CCD camera to image the whole area of a 96 well plate. The image is analyzed to calculate the total fluorescence per well for all the material in the well. Similarly, Molecular Devices, Inc. (Sunnyvale, Calif.) describes a system (FLIPR) which uses low angle laser scanning illumination and a mask to selectively excite fluorescence within approximately 200 microns of the bottoms of the wells in standard 96 well plates in order to reduce background when imaging cell monolayers. This system uses a CCD camera to image the whole area of the plate bottom. Although this system measures signals originating from a cell monolayer at the bottom of the well, the signal measured is averaged over the area of the well and is therefore still considered a measurement of the average response of a population of cells. The image is analyzed to calculate the total fluorescence per well for cell-based assays. Fluid delivery devices have also been incorporated into cell based screening systems, such as the FLIPR system, in order to initiate a response, which is then observed as a whole well population average response using a macro-imaging system.
However, cell populations are biologically heterogeneous, and the high spatial and temporal frequency of chemical and molecular information present within cells makes it impossible to extract high-content information from populations of cells using conventional techniques. Indeed, conventional techniques for monitoring and analyzing, for example, ion oscillations using fluorescence has substantial drawbacks; for example, photobleaching and lack of specific information concerning individual cells. Conventional techniques also are not fast or cost-efficient; for example, dose-dependent experiments using the patch-clamp technique typically require two days to complete.
In contrast to high throughput screens, high-content screens have also been developed to address the need for more detailed information about the temporal-spatial dynamics of cell constituents and processes. High-content screens automate the extraction of multicolor fluorescence information derived from specific fluorescence-based reagents incorporated into cells (Giuliano, K. A. and Taylor D. L., “Measurement and manipulation of cytoskeletal dynamics in living cells,” Curr Opin Cell Biol., 7(1):4-12 (1995)). Cells are analyzed using optical systems that can measure spatial and temporal dynamics (Farkas, D. L. et al., “Multimode light microscopy and the dynamics of molecules, cells, and tissues,” Ann. Rev. Physiol., 55:785-817 (1993)). With high-content screening, the concept is to treat each cell as a “well” that has spatial and temporal information on the activities of the labeled constituents. High-content screens can be performed on either fixed cells, using fluorescently labeled antibodies, biological ligands, and/or nucleic acid hybridization probes, or live cells using multicolor fluorescent indicators and “biosensors.” The choice of fixed or live cell screens depends on the specific cell-based assay required. Fixed cell assays provide an array of initially living cells in a microtiter plate format which can be treated with various compounds and doses being tested. Thereafter, cells can be fixed, labeled with specific reagents, measured and no environmental control of the cells is required after fixation. In this way, spatial information is acquired at one time point. Live cell assays provide an array of living cells containing the desired reagents which can be screened over time and space. Environmental control of the cells (temperature, humidity, and carbon dioxide) is required during measurement, since the physiological health of the cells must be maintained for multiple fluorescence measurements over time. Fluorescent physiological indicators and “biosensors” can report changes in biochemical and molecular activities within cells (Hahn et al., In Fluorescent and Luminescent Probes for Biological Activity, W. T. Mason (ed.), pp. 349-359 (1993) Academic Press, San Diego).
Scanning confocal microscope imaging (Go, W. Y. et al., “Quantitative dynamic multicompartmental analysis of cholecystokinin receptor movement in a living cell using dual fluorophores and reconstruction of confocal images,” Anal Biochem., 247(2):210-215 (1997)) and multiphoton microscope imaging (Denk, W. et al., “Two-photon laser scanning fluorescence microscopy,” Science, 248:73-6 (1990)) are well established methods for acquiring high resolution images of microscopic samples. These optical systems provide for shallow depth of focus, which allows features of limited axial extent to be resolved against the background. For example, it is possible to resolve internal cytoplasmic features of adherent cells from the features on the cell surface. Because scanning multiphoton imaging requires very short duration pulsed laser systems to achieve the high photon flux required, fluorescence lifetimes can also be measured in these systems (Lakowicz, J. R. et al., “Fluorescence lifetime imaging,” Anal Biochem., 202:316-330 (1992)), providing additional capability for different detection modes. However, these imaging methods are limited by the efficiency, photostability and toxicity of the fluorescence in the chosen system. Thus, there remains a need in the art for instrumentation and methods to directly measure ion oscillations of individual cells in a sample that results from dose-dependent administration of a compound.
One example of ion oscillation occurs in calcium (Ca2+) channels, which are generally found in many cells where, among other functions, they play important roles in signal transduction. In excitable cells, intracellular calcium supplies a maintained inward current for long depolarizing responses and serves as the link between depolarization and other intracellular signal transduction mechanisms. Like voltage-gated sodium channels, voltage-gated calcium channels have multiple resting, activated, and inactivated states.
Calcium channel antagonists are potent vasodilators and are widely used in the treatment of hypertension and angina pectoris. Clinically approved compounds in the United States include, for example, dihydropyridines (e.g., amlodipine, felodipine, nifedipine, nicardipine, isradipine, nimodipine); benzothiazepines (e.g., diltiazem), phenylalkylamines (e.g., verapamil); and diarylaminopropylamine ether (e.g., bepridil) (See, e.g., U.S. Pat. No. 6,897,305).
Endocrine cells, including gonadotroph, somatotroph, and corticotroph cells, exhibit baseline spontaneous calcium oscillations (BSCOs) in vitro as well as in their native environment (Bonnefont, X. et al., “Rhythmic bursts of calcium transients in acute anterior pituitary slices,” Endocrinology, 141(3):868-75 (2000); Kwiecien, R. et al., “Differential management of Ca2+ oscillations by anterior pituitary cells: a comparative overview,” Neuroendocrinology 68:135-151 (1998); Kaftan, E. J. et al., “Mitochondria shape hormonally induced cytoplasmic calcium oscillations and modulate exocytosis,” J Biol Chem 275:25465-25470 (2000); Schlegel, W. et al., “Oscillations of cytosolic Ca2+ in pituitary cells due to action potentials. Nature 329:719-721 (1987); Charles, A. C. et al., “L-type Ca2+ channels and K+ channels specifically modulate the frequency and amplitude of spontaneous Ca2+ oscillations and have distinct roles in prolactin release in GH3 cells,” J Biol Chem 274:7508-7515 (1999); Surprenant, A., “Correlation between electrical activity and ACTH/beta-endorphin secretion in mouse pituitary tumor cells,” J Cell Biol 95:559-566 (1982); Adler, M. et al., “Intracellular calcium and hormone secretion in clonal AtT-20/D16-16 anterior pituitary cells,” Cell Calcium 10:467-476 (1989); Thomas, P., and Smith, P. A., “Tetrabutylammonium: a selective blocker of the somatostatin-activated hyperpolarizing current in mouse AtT-20 corticotrophs,” Pflugers Arch 441:816-823 (2001); Fiekers, J. F., and Konopka, L. M., “Spontaneous transients of [Ca2+]i depend on external calcium and the activation of L-type voltage-gated calcium channels in a clonal pituitary cell line (AtT-20) of cultured mouse corticotropes,” Cell Calcium 19:327-336 (1996); Maturana, A. et al., “Spontaneous calcium oscillations control c-fos transcription via the serum response element in neuroendocrine cells. J Biol Chem 277:39713-39721 (2002)). Minor differences in baseline and stimulated calcium oscillation patterns have been noted between normal corticotroph and AtT-20 cells (Kwiecien 1998). Baseline spontaneous calcium oscillations may represent the sum of cellular calcium channels mediating replenishment and maintenance of calcium concentrations required for intact calcium dependent signaling pathways and cellular homeostasis (Gill, D. L., and Patterson, R. L., “Toward a consensus on the operation of receptor-induced calcium entry signals,” Sci STKE 2004:39 (2004)). Their importance for regulation of ACTH secretion has also been described (Kwiecien 1998; Adler 1989; Tse, A., and Lee, A. K., “Voltage-gated Ca2+ channels and intracellular Ca2+ release regulate exocytosis in identified rat corticotrophs,” J Physiol 528(1):79-90 (2000)). Understanding these oscillations and their underlying mechanisms is important for cellular physiology as well as screening for novel drug treatments (Berridge, M. J. et al., “The versatility and universality of calcium signaling,” Nat Rev Mol Cell Biol, 1(1):11-21 (2000)).
Calcium oscillations are caused by repetitive periodic release of calcium from internal stores and subsequent recharging. Generally, calcium release-activated calcium channels (CRAC) as well as arachidonate-regulated calcium channels (ARC) contribute to oscillations (Shuttleworth, T. J. and O. Mignen, “Calcium entry and the control of calcium oscillations,” Biochem Soc Trans, 31(5):916-9 (2003)). However, at low agonist concentrations, ARC channels dominate calcium oscillations. In contrast, at higher concentrations, depletion of calcium stores becomes more profound and activation of CRAC channels leads to constantly elevated levels.
The different types of calcium channels have been broadly categorized into four classes, L-, T-, N-, and P-type, distinguished by current kinetics, holding potential sensitivity and sensitivity to calcium channel agonists and antagonists. L-type calcium channel antagonists such as nimodipine block spontaneous oscillations in AtT-20 clonal pituitary cells (Fiekers, J. F. and L. M. Konopka, “Spontaneous transients of [Ca2+]i depend on external calcium and the activation of L-type voltage-gated calcium channels in a clonal pituitary cell line (AtT-20) of cultured mouse corticotropes,” Cell Calcium, 19(4):327-36 (1996)). In contrast, voltage-gated sodium channels are not involved in spontaneous calcium oscillations (Fiekers 1996). Moreover, while AtT-20 cells have been shown to have T-type and L-type channels, only L-type channel antagonists reversibly block oscillations (Fiekers 1996). Indeed, activation of L-type channels produces large, transient and sustained calcium oscillations (Fiekers 1996). Changes in oscillation patterns have also been studied in AtT-20 cells in response to norepinephrine and somatostatin (Adler, M. et al., “Intracellular calcium and hormone secretion in clonal AtT-20/D16-16 anterior pituitary cells,” Cell Calcium, 10(7):467-76 (1989)).
Calcium channels mediate the influx of Ca2+ into cells in response to changes in membrane potential and/or concentration gradient, and because of their central roles in ion homeostasis and in cell signaling events, these channels are involved in a wide variety of physiological activities; for example, muscle contraction, cardiovascular function, hormone and neurotransmitter secretion, and tissue growth and remodeling processes. Multiple types of calcium channels have been identified in mammalian cells from various tissues, including skeletal muscle, cardiac muscle, lung, smooth muscle and brain. Not surprisingly, calcium channels are recognized as important targets for drug therapy (See Nuccitelli, R., Methods in Cell Biology: A Practical Guide of the Study of Calcium in Living Cells, Vol. 40, Academic Press (1994); also U.S. Pat. No. 6,686,193). They are implicated in a variety of pathologic conditions, including, for example, essential hypertension, angina, congestive heart failure, arrythmias, migraine and pain.
The disclosures of all documents referred to throughout this application are incorporated herein by reference.
The foregoing examples of the related art and limitations related therewith are intended to be illustrative and not exclusive. Other limitations of the related art will become apparent to those of skill in the art upon a reading of the specification and a study of the drawings.